Correlations of FRMD7 gene mutations with ocular oscillations

Mutations in the FERM domain containing 7 (FRMD7) gene have been proven to be responsible for infantile nystagmus (IN). The purpose of this study is to investigate FRMD7 gene mutations in patients with IN, and to evaluate the nystagmus intensity among patients with and without FRMD7 mutations. The affected males were subdivided into three groups according to whether or not having FRMD7 mutations and the types of mutations. Fifty-two mutations were detected in FRMD7 in 56 pedigrees and 34 sporadic patients with IN, including 28 novel and 24 previous reported mutations. The novel identified mutations further expand the spectrum of FRMD7 mutations. The parameters of nystagmus intensity and the patients’ best corrected visual acuity were not statistically different among the patients with and without identified FRMD7 mutations, and also not different among patients with different mutant types. The FERM-C domain, whose amino acids are encoded by exons 7, 8 and 9, could be the harbor region for most mutations. Loss-of-function is suggested to be the common molecular mechanism for the X-linked infantile nystagmus.

Statistical analysis. Descriptive analysis was performed. Frequency (proportion) or mean +/− SD was reported for categorical or continuous variables, respectively. Analysis of Variance (ANOVA) was used to compare the difference among the patients with and without mutations. A P value of < 0.05 was considered statistically significant. No multiplicity adjustment was implemented. All statistical analyses were performed by R (version, 4.1.0).
All novel missense mutations identified in this study were predicted to be deleterious to the protein function and structure through online program analysis by using SIFT, PolyPhen-2, MutationTaster, CADD and PyMOL. The calculated scores for all mutations using the programs of SIFT, Polyphen2, CADD and Splice AI, as well as the ACMG classification, were listed in Table 1. A summarized description of PyMol results was listed in Table 2. As an example, the structure damage by M258T was shown in Fig. 3, and others were not shown here but may be supplied if requested.

Comparisons of parameters.
Affected males with or without FRMD7 mutations were collected to compare the difference between the best corrected visual acuity (BCVA) and nystagmus intensity because the affected males were hemizygote and carried only one affected allele on the X-chromosome. For comparison, the affected males were categorized into three subgroups. Group 1 (G1) consisted of 20 affected males with missense mutations; Group 2 (G2) included 18 affected males with premature translation termination; and Group 3 (G3) was composed of 19 affected males without FRMD7 mutations. The BCVA was evaluated for all patients in three groups. However, eye movement recording was obtained 14 in G1, 13 in G2 and 13 in G3. The mean of BCVA was 0.56 ± 0.14 in G1, 0.55 ± 0.15 in G2, and 0.52 ± 0.15 in G3 (Fig. 4). There were no significant differences in BCVA among these three groups (P = 0.474, P > 0.05).
The waveforms of nystagmus were varied as pendular, pendular with foveating saccades, jerk, jerk with extended foveation, as well as dual jerk waveforms recorded in our patients. No specific waveforms were www.nature.com/scientificreports/ associated with a specific mutant type. Even within a pedigree, waveforms were varied in the patients and their relatives, such as in a pedigree with a mutation of c.773T > C (p.M258T) (Fig. 3D,E). Eye movement recordings showed that the amplitude of oscillation was on the average of 3.20° ± 1.93°in G1, 3.44° ± 2.53° in G2, and 3.29° ± 2.39°in G3 at the primary gaze position (Figs. 5, 6). There were no significant differences among these three groups in the amplitude (P = 0.967, P > 0.05). While the frequency of oscillation was a mean of 2.97 Hz ± 1.35 Hz in G1, 2.99 Hz ± 1.92 Hz in G2, and 3.64 Hz ± 2.24 Hz in G3 at the primary gaze position. There were no significant differences among these three groups in the frequency (P = 0.609, P > 0.05).

Discussion
In this study, we found 52 mutations in FRMD7 from 56 Chinese pedigrees and 34 sporadic patients with IN, including 28 novel and 24 known mutations. To our knowledge, the number of mutations detected in this study is the largest in a series of reports about investigating FRMD7 mutations in the patients with IN. The mutation detection rate is higher in the X-linked pedigrees (46/56) than that in the sporadic patients (6/34). We suggest that the lower detection rate in the sporadic patients could be related to uncertainties in the inheritance modes, which might include new dominant or recessive inheritance patterns. This would be consistent with previous findings, which show that the X-linked mode accounts for 90% IN patients with family history, the autosomal dominant mode accounts for about 10%, and the autosomal recessive pattern is exceedingly rare.
Recurrent mutations and mutations at the same codon are also found in the patients in different families. However, recurrent mutations have been proven to be independently from a different chromosomal background rather than a common founder through haplotype analysis. As shown in Table 2, c.70G > T (p.G24W) and c.70G > A (p.G24R) represent a change from a highly conserved small neutral amino acid to larger hydrophobic polar and positively charged amino acids, respectively. These are not conservative in terms of their physical characteristics as estimated using a Grantham formula 12 . Similarly, c.887G > C (p.G296R), c.887G > A p.G296D),  www.nature.com/scientificreports/ and c.887G > T (p.G296V) represent changes from a highly conserved very small, neutral amino acid to larger positively charged, negatively charged hydrophilic, and aliphatic hydrophobic amino acids, respectively, also distant on the Grantham chart. These would be expected to disrupt the local environment and structure of the FRMD7 protein with subsequent interference with its function. FRMD7 encodes a protein with 714 amino acids belonging to the FERM-domain family members due to four proteins of the protein 4.1, ezrin, radixin and moesin in its structure 13 . As in most other FERM-domain family members, the highly conserved N-terminus consists of three domains of FERM-N, FERM-M, and FERM-C which are responsible for localizing the proteins to the plasma membrane 14,15 . The FERM-adjacent (FA) domain is located in the middle of the N-terminus and C-terminus of FRMD7 protein, playing a role in regulation of protein function through modifications such as phosphorylation. The C-terminus of FRMD7 has no significant homology with other proteins 15 . FRMD7 is highly expressed in the retina and midbrain where the center of ocular movement is located, and plays an important role in neurite development as well as in the control of eye movement and gaze stability 5,13 .
Structurally, the FERM-C domain is the third domain within the FERM domain, and constituted mainly by amino acids encoded from exon 7-9. This domain has many specific protein binding sites, playing important roles in linking the membrane with cytoskeleton as well as transmitting signals [17][18][19] . Recently, FRMD 7 has been found to bind to CASK and GABRA2. As a member of the membrane-associated guanylate kinase (MAGUK) family, CASK is found to be involved in regulation of neurite outgrowth and formation of dendritic spines. FRMD7 mutations would disrupt the interaction with CASK, impairing CASK-induced neurite formation and affecting neural and retinal development 13 . Clinically, hypoplasia of fovea and optic nerve have been detected from patients with FRMD7 mutations 20 . Thus, one of possible mechanisms of nystagmus could be related to the arrested neural and retinal development during the early embryonic phase 21 . Another possible mechanism Wild-type L57 is located in ß -folding region. The molecular quantity of mutant-type R57 is increased, the polarity is changed from non-polar to positively charged, and from neutral to alkaline. Mutant-type R57 will bring the strong basic amino acid residue I63 into the hydrophobic region, and destroy protein stability The molecular quantity of mutant-type Y85 is increased, the polarity is changed from negatively charged to uncharged. Y85 loses interaction with G87 3 c.367T > C p.S123P Wild-type S123 is located in the a-helix region. The molecular quantity of mutant-type P123 is increased, the polarity is changed from uncharged polar to non-polar. P123 loses interaction with A119, L120, and I158 4 c.586G > T p.D196Y The molecular quantity of mutant-type Y196 is increased, the polarity is changed from negatively charged to uncharged. Y196 loses interaction with S195 and E198 5 c.616G > A p.V206I The molecular quantity of mutant-type I206 is increased The molecular quantity of mutant-type R210 is increased, the polarity is changed from uncharged to positively charged 7 c.686G > A p.R229H Wild-type R229 is replaced by mutant-type H229, which interacts with K241 by two hydrogen bonds. The molecular quantity of mutant H229 is decreased Wild-type M258 interacts with amino acid residues K236 and P193. The molecular quantity of mutanttype T258 is decreased, the polarity is changed from non-polar to uncharged polar. Mutant-type T258 loses interaction with P193 and increases interaction with S260. S260 is a phosphorylation site. T258 affects FRMD7 regulation through phosphorylation 9 c.849G > C p.E283D The molecular quantity of mutant-type D283 is decreased. D283 increases interaction with K285 10 c.887G > C p.G296R Wild-type G296 and the adjacent amino acid residue S294 form the ß -folded region. The molecular quantity of mutant-type R296 is increased, the polarity is changed from uncharged polar to positively charged. R296 increases interaction with S298 11 c.887G > A p.G296D Wild-type G296 and the adjacent amino acid residue S294 form the ß -folded region. The molecular quantity of mutant-type D296 is increased, the polarity is changed from uncharged to negatively charged.D296 loses interaction with S294 and increases interaction with S297 12 c.887G > T p.G296V Wild-type G296 and the adjacent amino acid residue S294 form the ß -folded region. The molecular quantity of mutant-type V296 is increased, the polarity is changed from uncharged polar to non-polar. www.nature.com/scientificreports/       (Fig. 8). Others are nonsense, splicing and indel mutations which would produce a premature terminate codon and generate either a truncated protein or an abnormal mRNA that would be degraded due to the nonsense-mediated decay mechanism. The previous study showed that the severity of IN was highly associated with the amount and the location of the expressed protein 13 . Missense mutants with lower expression in the cytoplasm or localized aberrantly to the nucleus would produce a severe clinical feature due to the dominant-negative mechanism, while mutants caused by premature terminated translation would be possible to produce a much severer clinical phenotype, because mutants would be restricted to the nucleus 13 .
Our data does not support the correlation between the genotype and the phenotype in IN. According to the previous study, the affected males carrying with premature terminated translation would have much severer clinical features than those males who carry with missense mutations. However, there is no statistical difference between them on their corrected visual acuity, and on nystagmus amplitude and frequency. As far as the visual acuity is concerned, even though among the patients in a pedigree carrying with a common mutation, patients have variable visual acuity. For example, in the pedigree with a missense mutation of c.812G > T (p.C271F), patients had variable visual acuity from 0.2 to 1.0, while in the pedigree carrying with mutation of c.689-690delAG (p.S232Ffs*2), the visual acuity varied from 0.4 to 1.0 in the patients. Because the female carriers usually do not have symptoms, we think that loss-of-function could be the common molecular mechanism for the X-linked infantile nystagmus, no matter what kinds of mutations they carry.

Conclusions
We identify 28 novel mutations in the FRMD7 gene which would further expand the spectrum of FRMD7 mutations. The mutation hotspot is located at the FERM-C domain and is clustered in exon 7-9. The intensity of oscillations is not determined by the types of FRMD7 gene mutations, and not determined by absence or presence of mutations. Loss-of-function could be the common molecular mechanism for the X-linked infantile nystagmus.   www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.